The present invention relates to the expression of mitochondrial P450 in yeast. The mitochondrial P450 includes a chimeric P450 in which a signal sequence present at the N-terminus of a mammalian mitochondrial P450 has been substituted by a signal of a yeast mitochondrial protein and a chimeric P450 in which the latter signal has been further substituted by a targeting signal sequence to microsome. More specifically, the present invention relates to genes which encode a rat liver mitochondria P450.sub.c25 precursor, a chimeric P450 in which a matured P450.sub.c25 is connected with 29 amino acid residues which is believed to be the mitochondrial targeting signal of the yeast cytochrome c oxidase subunit IV (COXIV), and a chimeric P450 in which a signal sequence comprising 15 amino acid residues at the N-terminus of bovine adrenal P450.sub.17 .alpha. which is believed to be the microsomal targeting signal is followed by a matured P450.sub.c25, respectively.
The present invention further relates to an expression plasmid for producing the enzyme in a large scale, a recombinant yeast strain carrying the expression plasmid, a process for producing the enzyme by the use of the recombinant yeast, and a process for producing 58-cholestane-3.alpha.,7.alpha.,12.alpha.,27-tetrol (referred to hereinafter as TeHC), 25-hydroxy-vitamin D.sub.3 and 1.alpha.,25-dihydroxyvitamin D.sub.3.
The term chimeric P450 used in this specification means hereinafter P450 comprising a foreign N-terminal targeting signal sequence and a matured mitochondrial P450 at C-terminus.
P450 is a hemoprotein existing widely in biological fields from microorganisms to mammals and catalyzes as a terminal enzyme of electron transport chains monooxygenation toward a variety of lipophilic compounds as substrates.
P450 as the terminal enzyme in electron transport chains has a variety of molecular forms, which exhibit different substrate specificities and thus can catalyze the hydroxylation of a wide variety of lipophilic compounds. P450-dependent electrontransport chains in mammals are classified into two groups; microsomal and mitochondrial types.
In microsomes, NADPH-cytochrome P450 reductase (reductase) containing flavin adenine dinucleotide and flavin mononucleotide as cofactors in the molecule transfers electrons from NADPH to P450. In mitochondria, NADPH-ferredoxin reductase containing flavin adenine dinucleotide as a cofactor in the molecule and ferredoxin containing non-heme iron as a cofactor in the molecule transfer electrons from NADPH to P450.
The present inventors have already succeeded in industrially useful hydroxylations by producing several microsomal P450s and reductases in yeast and using their recombinant yeast strains.
That is, yeast strains producing the enzymes have been obtained by isolating genes for rat liver P450c, bovine adrenal P450.sub.17 .alpha. and P450.sub.c21, respectively, making expression plasmids containing these genes, respectively, and transforming yeast with the expression plasmids (Japanese Patent Kokai (Laid-Open) Nos. 56072/1986, 47380/1989 and 31680/1990).
These yeast strains exhibited monooxygenase activities depending on P450 molecular forms produced therein, respectively.
The present inventors have also succeeded in the expression of the enzyme having P450 reducing ability within yeast by isolating rat liver reductase gene or yeast reductase gene (Japanese Patent Kokai (Laid-Open) Nos. 19085/1987 and 51525/1990).
Moreover, the present inventors have succeeded in creating a yeast strain which produces both P450 and reductase (Japanese Patent Kokai (Laid-Open) No. 104582/1987) and novel monooxygenases having the functions of both enzymes (Japanese Patent Kokai (Laid-Open) Nos. 44888/1988 and 23870/1990, and Japanese patent Application No. 71250/1989).
By these techniques, the present inventors have successfully produced acetaminophene or steroid hormone intermediates useful as medicines with these P450-producing yeast strains.
On the other hand, the mitochondrial P450 participates in many biological reactions which synthesize physiologically important compounds such as active vitamin D.sub.3 and steroid hormones, and thus the creation of a yeast strain producing the mitochondrial P450 molecular form has a high industrial applicability.
However, there has not hitherto been reported the expression of an active mitochondrial P450 in yeast.
Furthermore, two enzymes, ferredoxin and ferredoxin reductase, are required in addition of P450 for the expression of mitochondorial P450-dependent monooxygenase activity.
Thus, the present inventors have tried to create a bioreactor for the expression of the mitochondorial P450 in yeast and of the mitochondrial P450-dependent monooxygenase activity.
Rat liver P450.sub.c25 which is a mitochondrial P450 is synthesized as a precursor comprising 533 amino acids in cytoplasm and then transported to mitochondria. During these processes, 32 amino acids at the amino terminus acting as a mitochondrial targeting signal are removed to yield a matured P450.sub.c25 comprising 501 amino acids (molecular weight: 57 KD) on the mitochondrial membrane.
The present inventors have already found that when a bovine adrenal mitochondrial protein is expressed in yeast, the substitution of its signal peptide part by that of yeast cytochrome c oxidase subunit IV (referred to hereinafter as COXIV) increases the expression amount remarkably (Japanese Patent Application No. 136496/1990),